It is estimated that over 75% of human bacterial infections are biofilm-related, yet relatively little is known about the biochemical mechanisms that govern biofilm formation. The development of a greater understanding of biofilm physiology therefore has profound implications for both basic bacteriology, and biomedical applications. In particular, the discovery of small molecule inhibitors of biofilm formation provides an opportunity to create a set of target-independent tools for delineating the complex regulatory cascade that control biofilm formation, and to develop new therapeutics for biofilm-related infections. We have recently developed an image-based, high content screening approach for visualizing biofilm inhibitors under high-throughput conditions. Application of this screening technology to the National Cancer Institute screening libraries has shown that this tool can be used to effectively identify biofilm inhibitors form small molecule screening libraries. Within the scope o this grant, we aim to further refine and optimize this screening platform to include the human pathogen Pseudomonas aeruginosa, and to identify those compounds that selectively impact cyclic-di-GMP signaling pathways; a major regulatory mechanism in biofilm formation. Secondly, we aim to explore the modes of action of these probes through a suite of analyses, including confocal microscopy, functional genomics and molecular genetics. We further aim to examine the effect on bacterial viability of co-dosing biofilm inhibitors with conventional antibiotic therapies to explore their utility as combination therapies for treatment of bacterial biofilm infections. Finally, we will develop sustainable methods of production of lead compounds through fermentation optimization studies to prepare lead candidate projects for pre-clinical development. PUBLIC HEALTH RELEVANCE: Bacterial infections remain one of the leading causes of hospitalization in the developed world. In many instances, these infections are made more difficult to treat by the presence of bacterial biofilms. Biofilms are layers of bacterial 'slime', which coat both biological surfaces (such as the lining of the lung) and artificial surfaces (such as stents and catheters). Biofilms create a dense matrix of proteins and sugars, and protect the bacteria from the action of antibiotics. This project aims to find small molecules that disrupt the formation of biofilms, and to use these compounds to better understand the mechanisms by which biofilms are formed, using state-of-the-art fluorescence imaging microscopy and small molecule chemical libraries.